There is a continuous need in medical practice, research and diagnostic procedures for rapid, accurate and qualitative or quantitative determinations of biological substances which are present in biological fluids at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, vaginal secretions, dental plaque, gingival crevicular fluid and other biological specimens has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment.
To provide such determinations, various methods have been devised for isolating and identifying biological substances employing specific binding reactions between the substance to be detected (sometimes identified as a "ligand") and a compound specifically reactive with that substance (sometimes identified as a "receptor").
After the formation of a specific binding complex between ligand and receptor, it is usually necessary to separate the complex from uncomplexed materials. A most common specific binding reaction involves an antigen (or antigen-like material) and its corresponding antibody which form an immunological complex. Uncomplexed antigen and antibodies are generally separated from the complex using various techniques. For example, separation may be accomplished by filtration, centrifugation or affinity chromatography. However, in most assays, the complex is solubilized and uncomplexed materials are washed from it. Common wash solutions include distilled water, various buffers and a number of solutions containing nonionic, anionic or cationic surfactants.
It has been found, however, that not every wash solution will effectively remove uncomplexed materials in every type of assay for a specific ligand. By effectiveness is meant that the uncomplexed materials are removed to such an extent that the assay result reflects only the presence of complexed materials with minimal background or cross-reactivity between the ligand and non-specific binding proteins. Thus, what may be useful as a wash composition for one assay is not necessarily useful in another. If the wash composition is ineffective, unwanted background may be present thereby obscuring the true assay result. One undesired result would be "false positives" from too high background or cross-reactivity. Another undesired result would be a true positive, but the background could obscure the magnitude of the positive result.
Specific microorganisms have been implicated as indicators for a number of periodontal diseases in humans and animals, such as gingivitis and periodontitis. The importance of such diseases is growing in the human population, especially as people live longer, and prevention of such diseases is becoming of considerable importance to dentists, insurance carriers and the health industry in general. In addition, proper dental care for animals is a growing concern in our culture.
Detection of microorganisms associated with periodontal diseases has been accomplished using culture techniques, DNA probes and a number of immunological procedures, such as agglutination assays, enzyme linked immunosorbent assays (ELISA) and others known in the art. ELISA utilizes the reaction of an extracted antigen from the microorganism(s) and the corresponding antibody to form an immunological complex. As noted above, usually uncomplexed materials are washed from the complex in order to provide an accurate assay result.
An advance in the art in the detection of microorganisms associated with periodontal diseases is described and claimed in the pending application U.S. Ser. No. 468,392 (filed Jan. 22, 1990 by Snyder). This case describes the simultaneous detection and differentiation of these microorganisms, and particularly Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia, in an immunometric (also known as "sandwich") assay using water-insoluble reagents in defined regions of a microporous filtration membrane. During the assay, uncomplexed materials were washed through the membrane using a common wash solution of sodium decyl sulfate in water (pH 7).
While the noted simultaneous assay represents an important advance in the art for detecting the noted microorganisms, in some cases, false positives were observed when a specimen containing high levels of one or more of the three microorganisms was contacted with the different regions of the membrane substrate containing the antibody reagents. A solution to this problem is critical since it is highly important for the user of the assay to discriminate among the microorganisms for effective diagnosis and treatment of disease without significant apparent cross-reactivity between an antigen and non-specific antibodies.